Langerhans cells (LCs) are immature dendritic cell (DC) subsets that perform two key surveillancetasks (antigen sampling and detection of danger signals) at the environmental interface. Our objective is to define behavioral mechanisms by which LCs achieve these tasks in their natural habitat (i.e., skin). For this purpose, we have developed novel imaging systems to acquire 4D images of LCs in living mice. In standard models of LC maturation, LCs exhibited dSEARCH motion (characterized by extension and retraction of dendrites), amoeba-like lateral migration, vertical migration, and LC-LC contact formation. Our aims are: J_) To define steady-state LC behaviors. LCs will be visualized in the l-Ap-EGFP knock-in mice (in which EGFP fluorescent signals are expressed by all DC subsets) and in the langerin-EGFP knock-in mice (in which only LCs express EGFP). We will record 3D images of EGFP* LCs every 2 min for 4 h by "time-lapse" multi- photon laser scanning microscopy in anesthetized mice to measure spaciotemporal kinetics of individual motile activities of EGFP+ LCs. We will study steady-state LC turnover by acquiring images of EGFP+ LCs in the same microscopic fields at different time points ("intermittent" imaging). By comparing the cellular movement recorded in the l-Ap-EGFP knock-in versus langerin-EGFP knock-in mice, we will assess the behaviors of other DC subsets in the skin. 2) To identify LC behavioral responses to pathological stimuli. We will study the turnover and motile activities of EGFP+ LCs after topical DNFB application, UVB radiation, and skin infection with Group A streptococci (GAS).The underlying mechanisms will then be assessed by locally injecting antagonists of selected cytokines, chemokines, and adhesion molecules and by back-crossing the EGFP knock-in mice with mutant mice deficient in target genes. 3) To develop intravital "multi-color" imaging systems. We will monitor dynamic processes in which EGFP* LCs internalize GAS strains engineered to expressing a red fluorescent protein (dsRed). The EGFP knock-in mice will be cross-bred with: a) IL-1p promoter-driven dsRed-transgenic mice to study LC maturation (red signals) and LC motility (green signals) independently, and b) K14 promoter-driven dsRed-transgenic mice to study dynamic interactions of LCs (green) with neighboring keratinocytes (red). Our study should provide important insights into the behavioral biology of LCs and the pathophysiology of skin disease in which LCs play pathogenic or protective roles.